Jo Bright

ABA‐induced NO generation and stomatal closure in Arabidopsis are dependent on H2O2 synthesis

Nitric oxide (NO) and hydrogen peroxide (H2 O2 ) are key signalling molecules produced in response to various stimuli and involved in a diverse range of plant signal transduction processes. Nitric oxide and H2 O2 have been identified as essential components of the complex signalling network inducing stomatal closure in response to the phytohormone abscisic acid (ABA). A close inter-relationship exists between ABA and the spatial and temporal production and action of both NO and H2 O2 in guard cells. This study shows that, in Arabidopsis thaliana guard cells, ABA-mediated NO generation is in fact dependent on ABA-induced H2 O2 production. Stomatal closure induced by H2 O2 is inhibited by the removal of NO with NO scavenger, and both ABA and H2 O2 stimulate guard cell NO synthesis. Conversely, NO-induced stomatal closure does not require H2 O2 synthesis nor does NO treatment induce H2 O2 production in guard cells. Tungstate inhibition of the NO-generating enzyme nitrate reductase (NR) attenuates NO production in response to nitrite in vitro and in response to H2 O2 and ABA in vivo. Genetic data demonstrate that NR is the major source of NO in guard cells in response to ABA-mediated H2 O2 synthesis. In the NR double mutant nia1, nia2 both ABA and H2 O2 fail to induce NO production or stomatal closure, but in the nitric oxide synthase deficient Atnos1 mutant, responses to H2 O2 are not impaired. Importantly, we show that in the NADPH oxidase deficient double mutant atrbohD/F, NO synthesis and stomatal closure to ABA are severely reduced, indicating that endogenous H2 O2 production induced by ABA is required for NO synthesis. In summary, our physiological and genetic data demonstrate a strong inter-relationship between ABA, endogenous H2 O2 and NO-induced stomatal closure.

Nitric oxide, stomatal closure, and abiotic stress

Various data indicate that nitric oxide (NO) is an endogenous signal in plants that mediates responses to several stimuli. Experimental evidence in support of such signalling roles for NO has been obtained via the application of NO, usually in the form of NO donors, via the measurement of endogenous NO, and through the manipulation of endogenous NO content by chemical and genetic means. Stomatal closure, initiated by abscisic acid (ABA), is effected through a complex symphony of intracellular signalling in which NO appears to be one component. Exogenous NO induces stomatal closure, ABA triggers NO generation, removal of NO by scavengers inhibits stomatal closure in response to ABA, and ABA-induced stomatal closure is reduced in mutants that are impaired in NO generation. The data indicate that ABA-induced guard cell NO generation requires both nitric oxide synthase-like activity and, in Arabidopsis, the NIA1 isoform of nitrate reductase (NR). NO stimulates mitogen-activated protein kinase (MAPK) activity and cGMP production. Both these NO-stimulated events are required for ABA-induced stomatal closure. ABA also stimulates the generation of H2O2 in guard cells, and pharmacological and genetic data demonstrate that NO accumulation in these cells is dependent on such production. Recent data have extended this model to maize mesophyll cells where the induction of antioxidant defences by water stress and ABA required the generation of H2O2 and NO and the activation of a MAPK. Published data suggest that drought and salinity induce NO generation which activates cellular processes that afford some protection against the oxidative stress associated with these conditions. Exogenous NO can also protect cells against oxidative stress. Thus, the data suggest an emerging model of stress responses in which ABA has several ameliorative functions. These include the rapid induction of stomatal closure to reduce transpirational water loss and the activation of antioxidant defences to combat oxidative stress. These are two processes that both involve NO as a key signalling intermediate.

A role for ETR1 in hydrogen peroxide signaling in stomatal guard cells

Signaling through the redox active molecule hydrogen peroxide (H2O2) is important for several processes in plants, such as stomatal closure, root growth, gravitropism, and responses to pathogen challenge ([Neill et al., 2002][1]; [Laloi et al., 2004][2]). Although oxidative modification of reactive

Differential requirement for NO during ABA-induced stomatal closure in turgid and wilted leaves.

Abscisic acid (ABA)-induced stomatal closure is mediated by a complex, guard cell signalling network involving nitric oxide (NO) as a key intermediate. However, there is a lack of information concerning the role of NO in the ABA-enhanced stomatal closure seen in dehydrated plants. The data herein demonstrate that, while nitrate reductase (NR)1-mediated NO generation is required for the ABA-induced closure of stomata in turgid leaves, it is not required for ABA-enhanced stomatal closure under conditions leading to rapid dehydration. The results also show that NO signalling in the guard cells of turgid leaves requires the ABA-signalling pathway to be both capable of function and active. The alignment of this NO signalling with guard cell Ca(2+)-dependent/independent ABA signalling is discussed. The data also highlight a physiological role for NO signalling in turgid leaves and show that stomatal closure during the light-to-dark transition requires NR1-mediated NO generation and signalling.

Pollen generates nitric oxide and nitrite: a possible link to pollen-induced allergic responses

Reactive nitrogen species (RNS), such as nitric oxide (NO), are ubiquitous and diverse signalling molecules involved in a wide range of physiological and pathophysiological processes in both animals and plants. Nitrite, a metabolite of NO turnover, has also been recently characterised as an important mediator of fundamental physiological mechanisms in mammalian cells, and is a substrate for NO production in several plant cell signalling processes. A previous study demonstrated that during plant reproductive processes, intracellular NO is produced by pollen, and that such NO could be important in signalling interactions between pollen and stigma. The aim of this study was to establish whether pollen releases NO and nitrite, using a wide range of plant species. Using a fluorimetric assay in conjunction with electron paramagnetic resonance (EPR) spectroscopy, the present study demonstrated that all hydrating pollen examined released NO, although some appeared to have more activity than others. Additionally, gas phase ozone-based chemiluminescence data showed that nitrite is also released from hydrating pollen. Given that pollen has interactions with other cells, for example in allergenic rhinitis (hay fever) in humans, it suggests that NO might be involved in mediating the responses of both plant and animal cells to pollen. These findings may have important implications for future allergy research, as it is possible that pollen-derived NO and nitrite may impact on mammalian cells during pollen-induced allergic responses.

Signaling on the stigma: Potential new roles for ROS and NO in plant cell signaling

Reactive oxygen species (ROS) and reactive nitrogen species, particularly NO, are key components of diverse signaling networks in animals and plants. We have recently shown that epidermal cells of stigmas from a range of different angiosperms accumulate relatively large amounts of ROS, principally H2O2, whereas pollen produces NO. Importantly, ROS/H2O2 levels appeared reduced in stigma cells supporting developing pollen grains compared to cells without pollen grains attached. To explore a possible link between pollen NO production and reduced levels of stigmatic ROS/H2O2, we supplied stigmas with NO and observed an overall reduction in levels of stigmatic ROS/H2O2. These new and unexpected data suggest a potential new signaling role for ROS/H2O2 and NO in pollen-stigma recognition processes.

Role of nitric oxide in regulating stomatal apertures

During stomatal closure, nitric oxide (NO) operates as one of the key intermediates in the complex, abscisic acid (ABA)-mediated, guard cell signaling network that regulates this process. However, data concerning the role of NO in stomatal closure that occurs in turgid vs. dehydrated plants is limited. The data presented demonstrate that, while there is a requirement for NO during the ABA-induced stomatal closure of turgid leaves, such a requirement does not exist for ABA-enhanced stomatal closure observed to occur during conditions of rapid dehydration. The data also indicate that the ABA signaling pathway must be both functional and to some degree activated for guard cell NO signaling to occur. These observations are in line with the idea that the effects of NO in guard cells are mediated via a Ca2+-dependent rather than a Ca2+-independent ABA signaling pathway. It appears that there is a role for NO in the fine tuning of the stomatal apertures of turgid leaves that occurs in response to fluctuations in the prevailing environment.

Nitric oxide in cell damage and protection

Less than ten years ago, the impact on plant biology of the gaseous free radical gas nitric oxide (NO.) related only to its toxic effects as a component of NOx, released into the atmosphere as an air pollutant during the combustion of fossil fuels. It is now clear that NO is a multi-faceted and versatile endogenous signalling molecule with an importance in many if not all aspects of plant growth and development. At least three enzymatic sources of NO in plants have been characterised and mechanisms that serve to scavenge NO have also been identified. Downstream signalling responses to NO include generation and action of the second messenger molecules calcium, cyclic GMP and cyclic ADPR, protein phosphorylation, protein nitrosylation and specific effects on gene expression. NO also interacts directly with Reactive Oxygen Species (ROS) and with components of ROS-activated signalling pathways. NO and ROS play key roles in an orchestra of plant defence responses. Rapid generation of NO and ROS following pathogen or elicitor challenge mediates a multitude of metabolic and transcriptional alterations including Programmed Cell Death (PCD). However, it is important to note that in some cases the actions of NO can be cytoprotective rather than toxic, potentially via antioxidant effects of NO. Furthermore prevention of NO synthesis or action can delay or inhibit PCD. In addition to the roles of NO and ROS in biotic stress responses, NO and ROS generation also occurs in response to various abiotic stresses, including UV radiation which itself can induce PCD. Recent data suggest that NO mediates some UV responses and that UV radiation can also stimulate the release of NOx from leaves. Key research questions to be addressed must be directed to the effects of UV on NO generation and action in plants. Research programmes will require methods to assess accurately NO emissions from leaves and other organs and to determine NO concentrations in cells and sub-cellular microdomains; the use of mutants and transgenic plants altered in NO synthetic and scavenging capacities; analyses of the molecular and biochemical events required for activation of PCD by NO and UV; and the development of techniques to monitor simultaneously cell death, NO and ROS generation in the field during exposure to UV.